How a holoenzyme for DNA replication is formed

Senthil K. Perumal, Wenhui Ren, Tae Hee Lee, Stephen J. Benkovic

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Loading of the phage T4 sliding clamp gp45 by the gp44/62 clamp loader onto DNA to form the holoenzyme and their disassembly pathways were investigated using FRET-based single-molecule and ensemble kinetic studies. gp44/62-mediated assembly of gp45 onto the DNA involves a rate-limiting conformational rearrangement of the gp45-gp44/62-DNA complex. Single-molecule measurements revealed the intermediates in gp45 loading and their interconversion, suggesting that the assembly is not concerted but is broken down into many small kinetic steps. Two populations of the gp45-gp44/62 -DNA complex are formed on the end-blocked DNA that are poised to form the holoenzyme with the polymerase. In the absence of a polymerase, the two clamp populations dissociated from the DNA along with gp44/62 with distinct rates. In the presence of polymerase, holoenzyme assembly involved the recruitment of the polymerase to the gp45-gp44/62-DNA complex mediated by the chaperoning activity of gp44/62. This transient multiprotein complex then decomposed through an ATP hydrolysis-dependent exit of gp44/62 leaving the holoenzyme on DNA. The rate of dissociation of the holoenzyme from the DNA is sensitive to whether the DNA ends are blocked, underscoring its mobility on the DNA.

Original languageEnglish (US)
Pages (from-to)99-104
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number1
DOIs
StatePublished - Jan 2 2013

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Holoenzymes
DNA Replication
DNA
Multiprotein Complexes
Bacteriophage T4
Population
Hydrolysis
Adenosine Triphosphate

All Science Journal Classification (ASJC) codes

  • General

Cite this

Perumal, Senthil K. ; Ren, Wenhui ; Lee, Tae Hee ; Benkovic, Stephen J. / How a holoenzyme for DNA replication is formed. In: Proceedings of the National Academy of Sciences of the United States of America. 2013 ; Vol. 110, No. 1. pp. 99-104.
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How a holoenzyme for DNA replication is formed. / Perumal, Senthil K.; Ren, Wenhui; Lee, Tae Hee; Benkovic, Stephen J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, No. 1, 02.01.2013, p. 99-104.

Research output: Contribution to journalArticle

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